RNA interference is being researched for use in fighting a slew of diseases, from eye disorders to HIV. Now Lyme disease has crawled onto the list: In the laboratory of Yale University School of Medicine professor Erol Fikrig, associate research scientist Sukanya Narasimhan is using RNAi to help develop a vaccine against Lyme disease and other deer tick-borne bacterial diseases.
Narasimhan has been researching ticks for about five years, and has been working towards the development of a vaccine against the spirochetes responsible for Lyme disease and various other disease-causing pathogens transmitted by the deer tick. Somewhere along the way, she told RNAi News, they decided to try to come up with a vaccine targeting the tick — namely its feeding practices — rather than the pathogen.
“If you can block tick feeding, we think we can block transmission of any of the pathogens that the tick harbors,” she said. “It is during feeding that the tick transmits the pathogens, so intuitively, one would expect that if you could block feeding, you could block pathogen transmission. That is an exciting possibility.”
According to Narasimhan, the deer tick lifecycle is dependent on blood meals at the three stages of the animal’s development: larval, nymph, and adult. “At every [stage of the] lifecycle, they take a blood meal, which lasts from six to eight days.” It is during the first 24 to 36 hours of this feeding that pathogens are transmitted to the host through the tick’s saliva, she said.
In order to successfully take a blood meal from a host, be it a rodent, deer, or a human, ticks need to produce anti-coagulants and anti-inflammatory agents in their saliva that block their host’s hemostatic pathways and immune responses, respectively. “These are the two key players” in the tick’s feeding process, Narasimhan said. “You can imagine that since they have to feed for six to eight days, they don’t come with one anti-coagulant and one anti-inflammatory — they come with an array … that might be targeting different steps of the [host’s] pathways at different times.”
As such, Narasimhan is using RNAi to discover which genes allow the deer tick to produce these anti-coagulant and anti-inflammatory agents. She is also looking for the genes that allow the pathogens to invade the tick’s salivary glands in the first place.
Already, Narasimhan and her colleagues have used RNAi to knock down the production of the salivary anti-coagulant Salp14 and its paralogs, finding that this reduced the ability of the deer tick to feed, as evidenced by a 50 to 70 percent decline in engorgement weights. These data were published in February in the Proceedings of the National Academy of Sciences.
For this work, Narasimhan injected into deer ticks double-stranded RNA made with the help of Ambion’s Megascript RNA synthesis kit. “We didn’t follow [Ambion’s] entire protocol because we already had our genes cloned into a vector that had T7 flanking the genes,” she noted. “For some parts of it we followed Ambion — especially for the T7 transcription step — but the annealing was a home-made recipe.”
Narasimhan noted that siRNAs had been evaluated, but they proved too expensive and less effective than dsRNAs. SiRNAs “were not as robust,” she said. Additionally, “with short-interfering RNAs, you have to try various constructs.
“We had tried only two or three different sequences,” she said. “If we had tried maybe 10, we would know which would be best, but since double-stranded RNA worked very well and it’s much less expensive,” those were used.
The use of RNAi to silence genes of interest, Narasimhan said, has proven to be not only effective but far simpler a task than previous approaches. “When we targeted a gene by making a recombinant protein and we immunized an animal, hoping that these antibodies would block the protein encoded by the gene, it wasn’t working as well” as RNAi, she said. “That’s because the proteins are glycosylated, so we weren’t able to mimic the [structure of the] in vivo protein when we made a recombinant protein. It was frustrating trying to find the function of these genes, leave alone finding a vaccine that would work.”
Encouraged by the Salp14 efforts and the overall efficacy of RNAi, Narasimhan and her colleagues are now working to screen a library of those genes that are expressed in deer ticks during the first 24 or so hours of feeding in order to “see which of these genes have a real function in feeding or [pathogen] transmission using RNA interference.” She said that she expects to identify about 50 genes that would appear crucial for feeding and make good candidates for RNAi knockdown.
Narasimhan said that she expects to have completed the first stage of the effort — coming up with a short list of genes — in the next three to four months. The timeline on a prophylactic vaccine for humans, however, is a bit longer, she said, especially since it is likely that a number of genes would need to be targeted to block tick feeding or pathogen transmission. A vaccine “might have to be a cocktail, and working out the right cocktail would involve some time and experimentation,” she said.
As for whether RNAi could become more than a useful tool for developing a vaccine, and be a vaccine itself, Narasimhan is not overly optimistic. “I think [a vaccine] would be protein or DNA[-based],” she said. “The RNA drug [field] is in its infancy, [but] maybe things will go well.”
— DM